Effect of Spaceflight and Spaceflight
Analogues on Microbial Virulence and
Infectious Disease Potential
Cheryl A. Nickerson, Ph.D.
Associate Professor
Goals of our Research:
Mitigate risk of infectious disease to crew
during human exploration of space to ensure
their health, safety, and performance
Critical to provide safe
passage for human
exploration to moon, Mars,
and beyond
New strategies
to combat
infectious
disease on Earth
Our Research
Interests
In-flight infections
Risk assessment/
Countermeasures
The pathogen
Low fluid shear culture
environment
Physiologically relevant
The host
Medically important
phenotypes not observed
during conventional culture
Vaccines/
Therapeutics
Infectious Disease Research and
Spaceflight
• Major advances in our knowledge about biological
systems have come by studying their responses to
extreme environments - (ex. temp, pH, etc) which
have led to major advances in global human health
breakthroughs
• Spaceflight is another extreme environment which
offers tremendous potential to provide new insight
into biological responses - including infectious
disease
• Spaceflight produces an environment that is
relevant to conditions encountered by the pathogen
during infection in the human host (low fluid shear)
WHY STUDY PATHOGEN RESPONSE TO SPACEFLIGHT?
9 Human presence in space accompanied by presence of microbes
9 Changes to microbes during spaceflight - potential negative impact on
health, safety, performance of crew
9 Certain Spaceflight conditions increase infectious disease risk:
• Increased mission duration
• Closed, self contained spacecraft
• Reliance on regenerative life support systems
• International crews
• Increased number/distribution of potential pathogens
• Immune system alterations
• Changes in antibiotic resistance
• Increased microbial virulence
9 Spaceflight/spaceflight analogue environments
biologically relevant - affect microbial physiology,
gene expression, pathogenesis - mechanism(s)
unclear
• New targets for vaccine/therapeutic development for
treatment/prevention
Pre-flight:
Ground Based Research
Our Model Intestinal Pathogen - Salmonella
typhimurium
• Common food-borne pathogen
>2,000,000 cases annually in U.S.
• A leading cause of death from
foodborne illness in U.S.
• Responsible for pre-flight ISS food
disqualification
• Gastroenteritis, secretory diarrhea in
healthy individuals
• Can cause serious/fatal infections in
immunocompromised
• Best characterized bacterial pathogen
Spaceflight analogue studies indicate
changes in microbial pathogen
characteristics
Salmonella typhimurium
Virulence
Gene
expression
* Classic virulence
genes down-regulated
* Ion response
genes/pathways!
Rotating Wall Vessel
bioreactor
Stress
Resistance
(Macrophage, acid, thermal,
osmotic, oxidative)
Nickerson et. al., 2000, Infect. Immun. 68:3147-3152; Wilson, et al., 2002, Proc. Natl. Acad. Sci. USA. 99:13807-13812;
Wilson, et al., 2002, Appl. Environ. Microbiol. 68:5408-5416; Nickerson, et al., 2004, Microbiol Mol Biol Rev, 68:345-361.
LSMMG increases Salmonella virulence
100
Percent survival
LSMMG
S. typhimurium
1xg
1xg
50
Mice
0
5
10
15
20
Time of Death (Days)
LSMMG
LSMMG
(pH=3.5)
S. typhimurium
S. typhimurium
LSMMG also increased
resistance of Salmonella to
osmotic and thermal stress
Macrophages
10
1xg
0
20
40
Time (minutes)
cell--associated
Number of cell
bacteria per well
Acid Stress
Percent survival
(log10)
100
60
106
105
104
103
20 min
LSMMG
1xg
Nickerson et. al., 2000, Infect. Immun. 68:3147-3152: Wilson et al., 2002, Applied and Environ Microbiol.
S. typhimurium
genome
The S. typhimurium
LSMMG regulon
163 genes
differentially
regulated by LSMMG
Propanediol utilization
Molydopterin binding
protein
Prolyl isomerase
Type III secretion
Amino acid/dipeptide
acid/dipeptide
transport
Ribosomal protein
subunits
LPS side chain synthesis
FeFe-S cluster formation
ABC transport apparatus
Stationary phase
induced
Transcriptional regulator
Other or unknown
function
1xg
LSMMG
Classic virulence genes down-regulated in
response to LSMMG
Nickerson et. al., 2000, Infect. Immun. 68:3147-3152; Wilson et. al., 2002, PNAS, 99(21): 13807 13812
Phenotypic Verification of Microarray Results:
LPS profiles of LSMMG- and 1xg-grown Salmonella
1xg
LSMMG
LPS
quantitation ratio
(LSMMG/1xg)
0.66 +/- 0.03
Wilson et. al., 2002, PNAS, 99(21): 13807-13812
Fur is a Regulator of the LSMMG response in Salmonella
WT
% survival (log10)
100
Fur - Global regulatory
LSMMG
10
1xg
protein senses Fe2+
levels, important for
Salmonella virulence
1
0 min
30 min
60 min
LSMMG
1xg
fur
% survival (log10)
100
LSMMG
10
1xg
1
0 min
30 min
LSMMG-induced acid
stress resistance is not
observed in S.
typhimurium fur mutant
60 min
Time
Wilson et. al., 2002, PNAS, 99(21): 13807-13812
Lessons from Ground Based
Studies
•
•
•
Microorganisms change
genotypic and phenotypic
characteristics, including
virulence
The cause is due to fluid
shear forces on the cell
surface
Ion types and
concentrations may
regulate this microbial
response
Importance for both Earth-based and Spaceflight
studies for countermeasures for infectious disease
MICROBE launches on
board Shuttle Atlantis
September 9, 2006
Astronaut Heidi Stefanyshyn-Piper
activates MICROBE on orbit
MDRV launches on board
Shuttle Endeavour
March 11, 2008
Commander Dom Gorie activates
MDRV on orbit
MICROBE Experimental Design
Effect of Spaceflight on Microbial Gene Expression and
Virulence
SPECIFIC AIMS:
1) Microarray and proteomic- mediated gene expression profiling of
S. typhimurium, P. aeruginosa, and C. albicans, in response to
spaceflight, as compared to normal gravity controls.
2) Determination of the effect of spaceflight on the virulence
potential of these microorganisms immediately following their return
from spaceflight.
* Better understand how microgravity affects genes, and may gain insight
into the genetic basis of how humans respond to microgravity
Model Microbes Chosen for this Study:
S. Typhimurium, P. aeruginosa, and C. albicans
Selection Criteria:
•Benchmark microbes for biological applications
•Isolated aboard Space Shuttle, Mir Space Station, ISS, or
their crew
•High risk potential for isolation aboard spacecraft
•May appear as a result of use of regenerative ALSS
•Availability of microarrays
•Previous ground-based studies demonstrating effect of
LSMMG on gene expression and virulence
MICROBE
Shuttle Atlantis, STS-115, launch Sept 9, 2006
Results:
Gene
expression
Spaceflight Globally
Alters Microbial Gene
Expression (167 genes)
Ion response pathways
Identification of global molecular
regulator (“master switch”) of spaceflight
induced cellular responses!
Disease
potential
Spaceflight Alters
Microbial Virulence
(Increased disease potential
for Salmonella in rich media)
In-flight
hardware
Cellular
morphology
Spaceflight alters
microbial morphology
(biofilm production)
* Synchronous ground controls maintained under identical conditions as those on-board Shuttle ground and in-flight hardware loaded with same sample.
Wilson et al., 2007, Proc. Natl. Acad. Sci. USA. 104(41):16299-304
Experimental hardware used in flight and in synchronous
ground controls at Kennedy Space Center (KSC)
flight
ground
*Synchronous ground controls maintained under identical conditions as those onboard Shuttle - ground and in-flight hardware loaded with same inoculum.
Wilson et al., 2007, Proc. Natl. Acad. Sci. USA. 104(41):16299-304
Spaceflight alters Salmonella morphology
In-flight grown S. typhimurium
showing presence of unidentified
extracellular matrix material
Synchronous ground-grown
S. typhimurium controls
Wilson et al., 2007, Proc. Natl. Acad. Sci. USA. 104(41):16299-304
Spaceflight Increases Salmonella Virulence in Murine Model of Infection
• Decreased time to death
• Increased percent mortality
• Decreased LD50 value
(cfu)
Flight
Ground
Wilson et al., 2007, Proc. Natl. Acad. Sci. USA. 104(41):16299-304
Spaceflight globally alters Salmonella gene expression
4500000
0
500000
4000000
• Protein secretion
Salmonella enterica
serovar Typhimurium
genome
3500000
• Outer membrane proteins
1000000
• Iron metabolism and storage
• Ion response pathways
4,857 kb
• Plasmid transfer functions
1500000
3000000
• Energy and metabolism
• Ribosomal proteins
2500000
2000000
• Small regulatory RNAs
• Biofilm formation
Global Proteomic Profiling
(MudPIT) identifies 73 proteins
differentially regulated by
spaceflight
• Transcriptional regulators
• Unknown function
Hfq - Master molecular
regulator identified
Wilson et al., 2007, Proc. Natl. Acad. Sci. USA. 104(41):16299-304
Hfq involvement confirmed with a ground-based
spaceflight analogue model (RWV)
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
Macrophage survival
6
5
Fold intracellular
bacterial replication
Acid survival ratio (1xg/LSMMG)
Acid Stress
4
3
2
1
0
WT
WT
hfq
Δhfq
hfq 3'Cm
hfq
3'Cm
hfq 3Cm g
hfq 3Cm L
delta hfq g
delta hfq L
invA
invA
Fur regulates LSMMG acid stress
resistance;
Hfq regulates Fur
1xg
LSMMG
1xg
hfq 3'Cm
LSMMG
Δhfq
Spaceflight data
confirmed with groundbased model
Wilson et. al., 2007, Proc. Natl. Acad. Sci. USA. 104(41):16299-16304.
The MDRV Flight Experiment
STS-123, March, 2008
A follow-up to MICROBE:
SPECIFIC AIMS:
• Independent validation of the effect of spaceflight on
Salmonella virulence in rich and minimal media
• Determine if modulation of different ion concentrations
can be used as novel way to counteract or block
spaceflight-associated increase in disease-causing
potential observed in Salmonella in rich media
• Determine common spaceflight responses that are
conserved/shared between different pathogenic
microbes
MDRV
Shuttle Endeavour, STS-123, launch March, 2008
Experiment Design:
Disease potential
• Independent validation of STS-115
results
• Test effect of media ion
composition
Media Composition
In-flight
hardware
*
Common response
conserved in other
pathogens?
Synchronous ground controls maintained under identical conditions as those on-board
Shuttle - ground and in-flight hardware loaded with same sample.
Manuscript Submitted
Lessons Learned from
Flight Studies
•
Spaceflight alters microbial genotypic and
phenotypic characteristics, including
virulence, in novel ways not observed
using traditional experimental approaches
•
Gene regulatory proteins identified during
spaceflight can be targeted for vaccine
development
•
Modulation of different ion concentrations
can be used to counteract/inhibit the
enhanced virulence of pathogens in
spaceflight
Importance for both Earth-based and spaceflight
studies for new strategies to combat infectious
disease
Acknowledgements
Arizona State University
James Wilson
Laura Quick
Emily Richter
Denise Fernandez
Lokesh Joshi
Phillip Stafford
Miti Shah
Michelle Kilcoyne
Joe Wang
Marc Porter
Randy Nelson
Roy Curtiss
University of Colorado HSC
Michael Schurr
NASA Johnson Space Center
Mark Ott
Duane Pierson
Mayra Nelman-Gonzalez
Don Tucker
Scott Smith
Sarah Zwart
Neal Pellis
Dennis Grounds
Heidi Stefanyshyn-Piper
and the crew of STS-115
Commander Dom Gorie and the
crew of STS-123
Oklahoma City University
Kent Buchanan
Tulane University HSC
Lisa Morici
Kerstin Honer zu Bentrup
NASA HQ
Jacob Cohen
Mark Uhran
NASA ARC
Steve Hing
Macarena Parra
Paula Dumars
Gary Jahns
Sid Sun
Russ Kerschmann
Kennedy Space Center
Ramona Bober
Kelly Norwood
Luke Catella
Jennifer Devich
Ashleigh Ruggles
Joseph Benjamin
Amber DeBaca
Satro Narayan
BioServe
Carla Goulart
Mark Rupert
Alex Hoehn
Louis Stodieck
University of Arizona
George Tsaprailis